This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Transthyretin (TTR) is a plasma carrier protein consisting of 127 amino acid residues. TTR normally exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR affect the stability of the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with the deposition of the TTR variants as amyloid fibrils in tissues and organs. TTR is also associated with senile systemic amyloidosis (SSA), also referred to as senile cardiac amyloidosis (SCA). SSA is a nonhereditary disorder that affects about 25% of individuals over 80 years old. In SSA, the amyloid fibrils are usually composed of wild type TTR and its fragments and are found mainly in the heart. We have found that approximately 85-95% of serum TTR is post-translationally modified by S-sulfonation and S-thiolation (conjugation with cysteine, cysteinyl glycine, and glutathione). Data obtained by ourselves and others indicates that S-Sulfonation of TTR decreases amyloid forming potential and possibly protects against protein aggregation and fibrillogenesis. The extent of thiol conjugation of proteins has been shown to increase with age. The purpose of this study was to evaluate the serum levels of S-sulfonated TTR in SSA patients seen at the BUSM Amyloid Treatment and Research Program, in an attempt to link a biochemical structural feature to disease pathogenesis. We originally hypothesized that increased S-sulfonation of transthyretin (TTR) at Cys10 and interactions with accessory proteins promote fibrillogenesis, and therefore act as effectors of senile systemic amyloidosis (SSA). To test this hypothesis, we analyzed and compared the relative abundance of S-sulfonated TTR isolated from the sera of patients with SSA, primary amyloidosis (AL) with cardiomyopathy, and non-amyloid controls. The results did not indicate that there is a direct link between TTR S-sulfonation and development of SSA. To examine the possibility that the profile of cysteine adducts in serum varies from that in the amyloid deposits, cardiac fibrils were extracted from autopsy tissues obtained on two separate cases of clinically diagnosed SSA. We developed methodology to isolate TTR from these tissues under non-reducing conditions in order to establish the degree of heterogeneity with respect to mass. We observed both truncation, dimerization and Cys-10 modification in the TTR fibrils. Top-down MS analysis enabled assignment of structures to truncated forms of the protein. We have published these results in Analytical Chemistry. We have carried out stability studies using fluorescently labelled recombinant forms of TTR and its two major metabolites, the S-sulfonated and S-cysteinylated forms and determined by analytical centrifugation that S-sulfonation indeed stabilizes the tetramer, whereas S-cysteinylation destabilizes it. A paper describing the results waspublished in J. Biol. Chem. Supplementary funding is now allowing evaluation of additional parameters that are associated with SSA, including other PTMs and the association of novel proteins.